Split-GFP tagging and live imaging of hair cell proteins

在过去的几十年里，毛细胞领域对毛细胞的内部工作有了很大的了解， 调节我们听觉和平衡感的感官感受器。多种因素的分子同一性 毛细胞发育和功能的重要性，以及它们对听力损失的意义已被阐明。 尽管取得了这些进展，但毛细胞的研究仍面临着许多挑战。尤其是活细胞成像和 蛋白质是细胞生物学研究的重要支柱，追踪蛋白质尤其困难，主要是因为 方法是对毛细胞进行转基因和培养。考虑到毛细胞功能的本质是微观的和 在纳米尺度的运动中，对蛋白质动力学性质的研究对于完整的 了解毛细胞的功能。因此，这项提议的目标是开发一种使成像 更容易了解活毛细胞中的蛋白质运动。 追踪蛋白质运动的黄金标准技术是标记蛋白质的活细胞成像。传统上， 这是通过表达融合到荧光蛋白的外源DNA构建物来实现的，这些外源DNA构建物通过 基因枪、电穿孔或病毒。转基因小鼠也已被采用。然而，这些方法， 可能会受到过度表达伪影和低转导/转染率的干扰。敲门造车 基因编码的荧光标记进入内源基因位点的(Ki)通过以下方式变得更容易处理 现代基因组编辑方法，但大DNA片段的Ki，如全长GFP编码序列， 仍然是困难的，并有可能影响基因表达。 为了应对这些挑战，我们计划开发一种鼠标工具包，以简化荧光标记 用于定位和活细胞成像的蛋白质。为此，我们采取了GFP分裂战略。在这两件事中- 在组分系统中，所关注的内源基因用一小部分GFP(“GFP 11”)进行遗传标记。 共表达GFP的剩余(非荧光)部分(“GFP 1-10”)重构了GFP荧光， 允许通过荧光显微镜进行成像。由于GFP11片段较小(48bps)，CRISPR- 介导KI进入内源基因座的效率很高。在初步研究中，我们证实了Split-GFP Approach忠实地概括了角质板蛋白LMO7的内源定位，通过传递 AAV将GFP 1-10包装到Lmo7-GFP 11 Ki小鼠毛细胞中。为了规避AAV介导的交付，我们 建议建立一个表达GFP1-10片段的转基因小鼠系。创始人们已经 已获得，并确认了生殖系遗传(SA1)。该系统将在实时成像上进行测试 难度增加的任务：在SA2中，我们将研究LMO7在毛细胞中的扩散动力学和运动。 在SA3中，挑战这个系统的灵敏度极限，我们将研究尖端连杆马达的动力学 发束中的复杂成分USH1C。

Over the past few decades, the hair cell field has gained great insight into the inner workings of hair cells, the sensory receptors that mediate our sense of hearing and balance. The molecular identity of many factors important for hair cell development and function, and their significance for hearing loss, have been elucidated. Despite this progress, the study of hair cells presents many challenges. In particular live-cell imaging and tracking of proteins, important pillars of cell biology studies, have been especially difficult, mainly due to limited means to transfect and culture hair cells. Considering that the essence of hair cell function is micro- and nanoscale movement, investigations of the dynamic properties of proteins is crucial for a complete understanding of hair cell function. The goal of this proposal therefore is to develop a tool that makes imaging of protein movements in living hair cells more accessible. The gold standard technique for tracking protein movement is live-cell imaging of tagged proteins. Traditionally, this is achieved by expression of exogenous DNA constructs fused to fluorescent proteins, delivered to cells by gene gun, electroporation or viruses. Transgenic mice also have been employed. These approaches, however, are potentially confounded by overexpression artifacts and low transduction/transfection efficiency. Knock-in (KI) of genetically-encoded fluorescent tags into the endogenous gene loci has become more tractable through modern genome editing methods, but KI of large DNA segments, such as the full-length GFP coding sequence, remains difficult and has the potential to affect gene expression. To address these challenges, we plan to develop a mouse tool kit that streamlines fluorescent tagging of proteins for localization and live cell imaging. To this end, we adopted the Split-GFP strategy. In this two- component system, the endogenous gene of interest is genetically tagged with a small part of GFP (“GFP11”). Co-expressing the remaining (non-fluorescent) portion of GFP (“GFP1-10”) reconstitutes GFP fluorescence, allowing imaging by fluorescence microscopy. Due to the small size of the GFP11 fragment (48 bps), CRISPR- mediated KI into the endogenous loci is highly efficient. In preliminary studies, we confirmed that the Split-GFP approach faithfully recapitulates the endogenous localization of the cuticular plate protein LMO7, by delivering AAV-packaged GFP1-10 into the hair cells of Lmo7-GFP11 KI mice. To circumvent AAV-mediated delivery, we propose to generate a transgenic mouse line that expresses the GFP1-10 fragment. Founders have already been obtained and germline transmission was confirmed (SA1). This system will be tested on live-imaging tasks of increasing difficulty: In SA2, we will study the diffusion kinetics and movement of LMO7 in the hair cell. In SA3, challenging the sensitivity limits of this system, we will investigate the dynamics of the tip link motor complex component USH1C in the hair bundle.